Efficient dual-negative selection for bacterial genome editing.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Gene editing is crucial for understanding gene function.
• Traditional bacterial genome modification methods like consecutive single-crossovers can be slow due to complex cloning and inefficient negative selection.
• Recombineering offers an alternative but can be difficult to implement in some species.

Purpose of the Study:

• To develop a more time-effective variant of consecutive single-crossovers for bacterial gene editing.
• To enhance the efficiency and applicability of bacterial genome manipulation.

Main Methods:

• Utilized rapid plasmid construction via Gibson assembly.
• Employed a convenient Escherichia coli donor strain.
• Implemented efficient dual-negative selection for improved suicide vector resolution.
• Generated in-frame deletions, insertions, and point mutations.

Main Results:

• Successfully generated various genetic modifications in Salmonella enterica with reduced hands-on time.
• Adapted the method for efficient gene editing in Pseudomonas aeruginosa.
• Achieved efficient gene editing in multi-drug resistant (MDR) Escherichia coli clinical isolates.

Conclusions:

• The developed method is time-effective and facilitates manipulation across multiple bacterial species.
• The technique is particularly useful for MDR clinical isolates.
• Anticipated broad applicability to other bacterial species, including those where recombineering is challenging.